B cells as a therapeutic target for IFN-ß in relapsing-remitting multiple sclerosis.

IFN-ß-1b is a first-line immunomodulatory therapy for relapsing-remitting multiple sclerosis (RR MS). However, its effects on B cells have not been characterized. In vitro studies of B cells derived from RR MS patients revealed that IFN-ß-1b decreases B cells' stimulatory capacity, as detected by inhibition of the Ag-specific T cell proliferative response upon Ag presentation by IFN-ß-1b-treated B cells. Our study has identified that IFN-ß-1b inhibited B cells' stimulatory capacity in RR MS patients and healthy controls through the suppression of CD40 and CD80 expression, whereas the MHC class I and II expression was not changed. IFN-ß-1b in vitro treatment inhibited B cell secretion of IL-1ß and IL-23 and induced IL-12 and IL-27. Supernatants transferred from IFN-ß-1b-treated B cells inhibited Th17 cell differentiation, as they suppressed gene expression of the retinoic acid-related orphan nuclear hormone receptor C and IL-17A and secretion of IL-17A. In addition, IFN-ß-1b induced B cells' IL-10 secretion, which may mediate their regulatory effect. Studies of B cells derived from RR MS patients treated with recombinant s.c. injected IFN-ß-1b revealed that they induced a significantly lower proliferative response in allogenic MLR than the B cells from untreated patients. Further confirming the IFN-ß-1b in vitro-induced changes in B cell cytokine secretion, B cells derived from the IFN-ß-1b-treated patients secreted significantly lower levels of IL-1ß and IL-23 and higher levels of IL-12 and IL-27 in comparison with the B cells derived from untreated patients. We conclude that IFN-ß-1b exerts its therapeutic effects in part by targeting B cells' functions that contribute to the autoimmune pathogenesis of RR MS.

Distinct hypoxia-induced translational profiles of embryonic and adult-derived macrophages.

Tissue resident macrophages are largely of embryonic (fetal liver) origin and long-lived, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation, such as hypoxia in the setting of myocardial ischemia. Prior transcriptome analyses identified BMDM and fetal liver-derived macrophage (FLDM) differences at the RNA expression level. Posttranscriptional regulation determining mRNA stability and translation rate may override transcriptional signals in response to hypoxia. We profiled differentially regulated BMDM and FLDM transcripts in response to hypoxia at the level of mRNA translation. Using a translating ribosome affinity purification (TRAP) assay and RNA-seq, we identified non-overlapping transcripts with increased translation rate in BMDM (Ly6e, vimentin, PF4) and FLDM (Ccl7, Ccl2) after hypoxia. We further identified hypoxia-induced transcripts within these subsets that are regulated by the RNA-binding protein HuR. These findings define translational differences in macrophage subset gene expression programs, highlighting potential therapeutic targets in ischemic myocardium.

IFN-beta inhibits human Th17 cell differentiation.

IFN-beta-1a has been used over the past 15 years as a primary therapy for relapsing-remitting multiple sclerosis (MS). However, the immunomodulatory mechanisms that provide a therapeutic effect against this CNS inflammatory disease are not yet completely elucidated. The effect of IFN-beta-1a on Th17 cells, which play a critical role in the development of the autoimmune response, has not been extensively studied in humans. We have investigated the effect of IFN-beta-1a on dendritic cells (DCs) and naive CD4(+)CD45RA(+) T cells derived from untreated MS patients and healthy controls in the context of Th17 cell differentiation. We report that IFN-beta-1a treatment down-regulated the expression of IL-1beta and IL-23p19 in DCs, whereas it induced the gene expression of IL-12p35 and IL-27p28. We propose that IFN-beta-1a-mediated up-regulation of the suppressor of cytokine signaling 3 expression, induced via STAT3 phosphorylation, mediates IL-1beta and IL-23 down-regulation, while IFN-beta-1a-induced STAT1 phosphorylation induces IL-27p28 expression. CD4(+)CD45RA(+) naive T cells cocultured with supernatants from IFN-beta-1a-treated DCs exhibited decreased gene expression of the Th17 cell markers retinoic acid-related orphan nuclear hormone receptor c (RORc), IL-17A, and IL-23R. A direct IFN-beta-1a treatment of CD45RA(+) T cells cultured in Th17-polarizing conditions also down-regulated RORc, IL-17A, and IL-23R, but up-regulated IL-10 gene expression. Studies of the mechanisms involved in the Th17 cell differentiation suggest that IFN-beta-1a inhibits IL-17 and induces IL-10 secretion via activated STAT1 and STAT3, respectively. IFN-beta's suppression of Th17 cell differentiation may represent its most relevant mechanism of selective suppression of the autoimmune response in MS.

T cell LFA-1-induced proinflammatory mRNA stabilization is mediated by the p38 pathway kinase MK2 in a process regulated by hnRNPs C, H1 and K.

Activation of the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1) in T cells induces stabilization of proinflammatory AU-rich element (ARE)-bearing mRNAs, by triggering the nuclear-to-cytoplasmic translocation of the mRNA-binding and -stabilizing protein HuR. However, the mechanism by which LFA-1 engagement controls HuR localization is not known. Here, we identify and characterize four key regulators of LFA-1-induced changes in HuR activity: the p38 pathway kinase MK2 and the constitutive nuclear proteins hnRNPs C, H1 and K. LFA-1 engagement results in rapid, sequential activation of p38 and MK2. Post-LFA-1 activation, MK2 inducibly associates with both hnRNPC and HuR, resulting in the dissociation of HuR from hnRNPs C, H1 and K. Freed from the three hnRNPs, HuR translocates from the nucleus to the cytoplasm, and mediates the stabilization of labile cytokine transcripts. Our results suggest that the modulation of T cell cytokine mRNA half-life is an intricate process that is negatively regulated by hnRNPs C, H1 and K and requires MK2 as a critical activator.

Cytokines and cardiovascular disease.

The role of cytokines in the pathogenesis of cardiovascular disease is increasingly evident since the identification of immune/inflammatory mechanisms in atherosclerosis and heart failure. In this review, we describe how innate and adaptive immune cascades trigger the release of cytokines and chemokines, resulting in the initiation and progression of atherosclerosis. We discuss how cytokines have direct and indirect effects on myocardial function. These include myocardial depressant effects of nitric oxide (NO) synthase-generated NO, as well as the biochemical effects of cytokine-stimulated arachidonic acid metabolites on cardiomyocytes. Cytokine influences on myocardial function are time-, concentration-, and subtype-specific. We provide a comprehensive review of these cytokine-mediated immune and inflammatory cascades implicated in the most common forms of cardiovascular disease.

Regulation of suppressors of cytokine signaling as a therapeutic approach in autoimmune diseases, with an emphasis on multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating, presumably autoimmune disease of the central nervous system (CNS). Among the available MS therapies, interferon (IFN)ß and the recently introduced statins have been reported to exert their immunomodulatory effects through the induction of SOCS1 and SOCS3 in various inflammatory cell subsets. The SOCS proteins negatively regulate cytokine and Toll-like receptors- (TLR-) induced signaling in the inflammatory cells. SOCS1 and SOCS3 have been reported to play an important role in the regulation of Th17-cell differentiation through their effects on the cells of the innate and adaptive immune systems. IFNß and statins inhibit Th17-cell differentiation directly and indirectly via induction of SOCS1 and SOCS3 expression in monocytes, dendritic cells (DCs), and B-cells. Due to their rapid induction and degradation, and SOCS-mediated regulation of multiple cytokine-signaling pathways, they represent an attractive therapeutic target in the autoimmune diseases, and particularly relapsing remitting (RR) MS.

Interferon-beta inhibits Th17 cell differentiation in patients with multiple sclerosis.

Interferon-beta (IFNbeta) has been used over the past 15 years as a first-line therapy for relapsing remitting multiple sclerosis (RR MS), however its mechanisms of action are still not completely elucidated. Recently discovered Th17 cells have been hypothesized to play a crucial role in the development of autoimmune diseases, including MS. Studies from our laboratory and others have demonstrated that IFNbeta treatment suppresses Th17 cells' differentiation, mediated by its effects on dendritic cells (DCs), B-cells and T-cells. IFNbeta induces the production of the Th17-suppressive cytokines interleukin (IL)-27 and IL-12 in DCs and B-cells through the phosphorylation of signal transducers and activators of the transcription protein (STAT)1. Its inhibition of the Th17-promoting cytokines IL-1b and IL-23 is mediated via induction of suppressor of cytokine signaling (SOCS)3 expression. In naïve CD45RA+ T-cells, IFNbeta directly suppresses Th17 cells' differentiation, as evidenced by the suppression of this cell subset's specific transcription factor retinoic acid-related orphan receptor (ROR)c, cytokine IL-17A and the surface markers chemokine receptor (CCR)6 and IL-23R. The IFNbeta-mediated induction of IL-10 in T-cells and B-cells represents an important additional immunoregulatory mechanism. Described IFNbeta's mechanisms of action selectively target Th17 cell-mediated autoimmune responses in patients with RR MS.

T cell LFA-1 engagement induces HuR-dependent cytokine mRNA stabilization through a Vav-1, Rac1/2, p38MAPK and MKK3 signaling cascade.

BACKGROUND: Engagement of the ß2 integrin, lymphocyte function-associated antigen-1 (LFA-1), results in stabilization of T cell mRNA transcripts containing AU-rich elements (AREs) by inducing rapid nuclear-to-cytosolic translocation of the RNA-stabilizing protein, HuR. However, little is known regarding integrin-induced signaling cascades that affect mRNA catabolism. This study examines the role of the GTPases, Rac 1 and Rac 2, and their downstream effectors, in the LFA-1-induced effects on mRNA. METHODOLOGY/PRINCIPAL FINDINGS: Engagement of LFA-1 to its ligand, ICAM-1, in human peripheral T cells resulted in rapid activation of Rac1 and Rac2. siRNA-mediated knockdown of either Rac1 or Rac2 prevented LFA-1-stimulated stabilization of the labile transcripts encoding IFN-γ and TNF-α, and integrin mediated IFN-γ mRNA stabilization was absent in T cells obtained from Rac2 gene-deleted mice. LFA-1 engagement-induced translocation of HuR and stabilization of TNF- α mRNA was lost in Jurkat cells deficient in the Rac guanine nucleotide exchange factor Vav-1 (J.Vav1). The transfection of J.Vav1 cells with constitutively active Rac1 or Rac2 stabilized a labile ß-globin reporter mRNA, in a HuR-dependent manner. Furthermore, LFA-1-mediated mRNA stabilization and HuR translocation in mouse splenic T cells was dependent on the phosphorylation of the mitogen-activated protein kinase kinase, MKK3, and its target MAP kinase p38MAPK, and lost in T cells obtained from MKK3 gene-deleted mice. CONCLUSIONS/SIGNIFICANCE: Collectively, these results demonstrate that LFA-1-induced stabilization of ARE-containing mRNAs in T cells is dependent on HuR, and occurs through the Vav-1, Rac1/2, MKK3 and p38MAPK signaling cascade. This pathway constitutes a molecular switch that enhances immune and pro-inflammatory gene expression in T cells undergoing adhesion at sites of activation and effector function.

Self-complementary AAV virus (scAAV) safe and long-term gene transfer in the trabecular meshwork of living rats and monkeys.

PURPOSE: AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals. METHODS: Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays. RESULTS: No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys. CONCLUSIONS: The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.

Simvastatin inhibits IL-17 secretion by targeting multiple IL-17-regulatory cytokines and by inhibiting the expression of IL-17 transcription factor RORC in CD4+ lymphocytes.

Statins, extensively used as cholesterol-lowering agents, have recently been identified as immunomodulatory agents. This study investigated the statins' mechanisms that target the autoimmune response in humans, and evaluated their therapeutic potential in multiple sclerosis. Our results demonstrated statin-mediated increases in suppressor of cytokine secretion (SOCS) 3 and suppressor of cytokine secretion 7, which negatively regulate the STAT/JAK signal transduction pathway and IL-6 and IL-23 gene expression in monocytes. Simvastatin also induced IFN-gamma, IL-4, and IL-27 production in monocytes, which together inhibited IL-17 transcription and secretion in CD4(+) T cells. IL-17-producing CD4(+) cells, referred to as Th17 cells, have recently been found to play a central role in the development of autoimmune diseases. Furthermore, simvastatin directly inhibited the expression of retinoic acid-related orphan nuclear hormone receptor C, a transcription factor that controls IL-17 production in CD4(+) T cells. This effect was reversed by mevalonic acid, a downstream metabolite of 3-hydroxy-3-methylglutaryl CoA reductase, confirming that simvastatin's specific effect is through the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase. These results provide evidence for the novel immunomodulatory mechanisms of statins, which selectively target the regulation of cytokine transcription involved in the development of the human autoimmune response. Based on the described immunomodulatory mechanisms, good safety profile and oral bioavailability, statins represent a promising therapeutic approach for multiple sclerosis and other chronic inflammatory diseases.